Targeted disruption of Cripto-1 in mice caused embryonic lethality at E7.5, whereas we unexpectedly found that ectopic Cripto-1 expression in mouse embryos also led to embryonic lethality, which prompted us to characterize the causes and mechanisms underlying embryonic death due to ectopic Cripto-1 expression. RCLG/EIIa-Cre embryos displayed complex phenotypes between embryonic day 14.5 (E14.5) and E17.5, including fatal hemorrhages (E14.5-E15.5), embryo resorption (E14.5-E17.5), pale body surface (E14.5-E16.5) and no abnormal appearance (E14.5-E16.5). Macroscopic and histological examination revealed that ectopic expression of Cripto-1 transgene in RCLG/EIIa-Cre embryos resulted in lethal cardiac defects, as evidenced by cardiac malformations, myocardial thinning, failed assembly of striated myofibrils and lack of heartbeat. In addition, Cripto-1 transgene activation beginning after E8.5 also caused the aforementioned lethal cardiac defects in mouse embryos. Furthermore, ectopic Cripto-1 expression in embryonic hearts reduced the expression of cardiac transcription factors, which is at least partially responsible for the aforementioned lethal cardiac defects. Our results suggest that hemorrhages and cardiac abnormalities are two important lethal factors in Cripto-1 transgenic mice. Taken together, these findings are the first to demonstrate that sustained Cripto-1 transgene expression after E11.5 causes fatal hemorrhages and lethal cardiac defects, leading to embryonic death at E14.5-17.5.To further determine whether or not Cripto-1 can initiate the mentioned-above tumors, heterozygous RCLG transgenic mice were first crossed to homozygous EIIa-Cre mice to generate RCLG/EIIa-Cre double transgenic mice, in which the Cripto-1 and Luc transgenes are simultaneously activated in a diffuse pattern. Additionally, because the efficiency of Cre-mediated recombination in RCLG/EIIa-Cre transgenic mice did not reach 100%, some cells in the RCLG/EIIa-Cre transgenic mice still harbored the mRFP gene. Therefore, mRFP and Luc expression were simultaneously detectable in the RCLG/EIIa-Cre transgenic mice through the use of whole-animal fluorescence and bioluminescence imaging.Unexpectedly, we failed to identify any Luc- and mRFP-positive mice from the 68 newborn progeny derived from mating heterozygous RCLG mice with homozygous EIIa-Cre mice, despite using in vivo fluorescence and bioluminescence imaging (Supplementary Figure S1A,B and Table S3) and PCR analysis (Supplementary Figure S1C). However, Luc- and mRFP-positive offspring were observed in the newborn progeny derived from mating RLG transgenic mice with homozygous EIIa-Cre mice (Supplementary Figure S2 and Table S3), suggesting that the both Cre/lox P system and the in vivo imaging system were functioning properly. Furthermore, both the Cripto-1 and Luc transgenes can be simultaneously “switched-on” in RCLG/EIIa-Cre transgenic mice (Fig. 1A). Based on the above findings, we hypothesized that the sustained expression of the Cripto-1 transgene during embryonic development can cause embryonic lethality.The wild-type FVB/N mice, the homozygous R26R reporter mice (B6; 129S4-Gt(ROSA)26Sortm1Sor/J)41, the homozygous EIIa-Cre transgenic mice (FVB/N-Tg(EIIa-Cre)C5379Lmgd/J)42 and the heterozygous hUb-CreERT2 mice [129S.Cg-Tg(UBC-cre/ESR1)1Ejb/J; Stock Number: 007179]22were obtained from the Model Animal Research Center of Nanjing University, China. The wild-type ICR mice were purchased from Cyagen Biosciences (Guangzhou) Inc., China. The EIIa-Cre mice42 carry a Cre transgene under the control of a zygotically expressed (EIIa-Cre) promoter that activates the expression of Cre recombinase in the early mouse embryo. EIIa-Cre-mediated recombination occurs in a wide range of tissues, including the germ cells that transmit the genetic alteration to progeny (http://jaxmice.jax.org/strain/003724.html). hUb-CreERT2 mice were generated through lentitransgenesis using a lentivirus that expresses the Cre-ERT2 from the human ubiquitin C promoter22. All of the animal care and experiments were performed according to the Study and Ethical Guidelines for Animal Care, Handling and Termination established by the Southern Medical University subcommittee on laboratory animal care. The present work was approved by the ethical committee of Southern Medical University and is covered by Chinese animal husbandry legislation.The authors thank all members of Xiao’s lab for their help in this project. We thank Dr. David S. Salomon (Center for Cancer Research, National Cancer Institute, USA) and Prof. Manuela Martins-Green (University of California) for generously providing pCI-Cripto-1 plasmid and pCAG-RLG plasmid, respectively. This work was supported by the National Natural Science Foundation of China (Grant No. 81172587, 81372896 and 81672689, to D. Xiao; Grant No. 81600488, to X.-L. Lin; Grant No. 81600086, to Y. Sun; Grant No. 61427807, to H,-F. Liu), the Natural Science Foundation of Guangdong Province of China (Grant No. 2014A030313294 to D. Xiao; Grant No. S2012010009212, to K. Xu), the Science and Technology Planning Project of Guangdong Province of China (Grant No. 2009B060300008 and 2013B060300013, to D. Xiao; Grant No. 2015A030302024, to X.-L. Lin), the China Postdoctoral Science Foundation (Grant No. 2015M572338 and Grant No. 2016T90792, to X.-L. Lin) and the Introduced Major Research and Development Project Funded by Fujian Province (Grant No. 2012I2004, to X.-D.Ma).Author Contributions This study was conceived, designed and supervised by D.X., Y.S., X.L.L. and W.Z. Experiments were performed by X.L.L., W.Z., J.J., T.L., G.X., S.W., X.L., Y.L., L.C., Y.Q., R.X., J.L., T.Z., W.C.H., B.C., Y.C. and K.X. Reagents/materials/analysis tools: W.H. The data were analyzed by D.X., Y.S., X.L.L. and W.Z. The general and administrative support was obtained from K.Y. The manuscript was written by D.X., Y.S. and X.L.L. and approved by all the authors.